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Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAs.

Yang Zhang1,2,3, Tuan M Nguyen1,2,4, Xiao-Ou Zhang5

  • 1Cancer Research Institute, Beth Israel Deaconess Cancer Center, Department of Medicine and Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02215, USA.

Genome Biology
|January 22, 2021
PubMed
Summary

CRISPR/Cas13d enhances circRNA silencing specificity, overcoming limitations of short hairpin RNAs (shRNAs). This breakthrough enables robust identification of circRNAs influencing hepatocellular carcinoma and drug resistance.

Keywords:
CRISPR/Cas13d systemHigh-throughput screeningcircRNAs

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Short hairpin RNAs (shRNAs) are commonly used for circRNA depletion via back-splicing junction targeting.
  • Existing shRNA methods show inconsistencies between circRNA knockdown and biological outcomes, raising concerns about their reliability.

Purpose of the Study:

  • To develop a more specific and robust method for circRNA silencing using CRISPR/Cas13d.
  • To identify functional circRNAs, including those associated with sorafenib resistance in hepatocellular carcinoma.

Main Methods:

  • Optimized single-guide RNA design for CRISPR/Cas13d targeting of circRNA back-splicing junction sites.
  • Parallel screening of shRNA and CRISPR/Cas13d libraries to validate specificity.
  • Utilized a CRISPR/Cas13d library for high-throughput screening of over 2500 hepatocellular carcinoma-related circRNAs.

Main Results:

  • CRISPR/Cas13d demonstrated markedly enhanced specificity in circRNA silencing compared to shRNAs.
  • Validated specificity through parallel screening experiments.
  • Identified a subset of circRNAs conferring resistance to sorafenib in hepatocellular carcinoma models.

Conclusions:

  • CRISPR/Cas13d offers a highly specific and effective tool for circRNA functional studies.
  • This technology facilitates high-throughput screening for identifying functionally relevant circRNAs, such as those involved in cancer drug resistance.